Method of inactivating viruses



United States Patent 1 3,031,378 METHOD OF INACTIVATING VIRUSES Morizo Ishidate, 608 Koenji 4-chome, Suginamiku,

Tokyo, Japan; Masakatsu Goto, 8-3 Yonbancho, Chiyodaku, Tokyo, Japan; and Kazuo Ogasawara, 1 Bikincho l-chome, Showaku, Nagoya, Japan No Drawing. Filed May 27, 1960, fier. No. 32,122

Claims priority, application Japan May 30, 1059 2 Claims. (Cl. 167-78) In general, formalin, phenol, merthiolate, etc. have been used as chemical substances for inactivated vaccines of bacteria and viruses. However, these substances .have been more or less toxic thereby exhibit toxicity to the host, 50 these chemicals are not desirable.

The present invention is based on a novel knowledge that, when certain concentration of glucuronolactone, glucuronic acid or its salt will be mixed with viruses in vitro, the viruses will be inactivated and also the antigenic properties of the viruses will not be so much reduced as by other known inactivating substances. The glucuronolactone, glucuronic acid or its salt, especially, sodium salt has hitherto been appreciated as a part of normal component present in the tissue of animals, so it does not exhibit any complication when administered to excess.-

Accordingly, the product obtained by the present invention can be advantageously used as improved vaccines.

The present invention shall now be explained in more details. A virus suspension is treated at the room temperature (20 C.) or a lower temperature with the addition of glucuronolactone, glucuronic acid or its salt to inactivate virus and then pH thereof is adjusted at 6-8. In this case, one must take care that the virus may not be inactivated by other factors as heat and the like.

The results of experiments in which mice inoculated with the virus suspensions treated by the application of the methods for the mouse hepatitis virus, Japanese B encephalitis virus, rabies virus and influenza virus are tabulated in the following.

EXAMPLE 1 10" dilution of mouse hepatitis virus (Busher strain, i.p., LD =10- /0.2 ml.) was prepared with a Bacto- Brain Heart Infusion Broth of Difco Laboratories. Three 10" dilutions are prepared with 25%, 12.5% and 6.25% sodium glucuronate adjusted pH 7.04 with phosphate buffer solution, and were maintained at the room temperature for one hour and then in an ice chest for 12 and 48 hours. 0.2 ml. of the solution was intraperitoneally inoculated in each mouse d dd strain 10 to 12 g.) Same procedure was performed with 10- and 10- dilutions of the mouse hepatitis virus. For controls, 10- and 10- virus suspensions were prepared with only phosphate butter solution.

The results for 10 days after inoculation of mixture virus and sodium glucuronate are given in Table 1.

Table 1 EXPERIMENTS ON INACTIVATION OF THE MOUSE HEPATIIIS VIRUS WITH SODIUM GLUCURONATE Concen- Concentration of the mouse hepatitis virus dilution tration of 1 Time of the sodileaviug um glu- 10'" 10' 10* in the ice curonate c est solution (4 0.) used in Mortality Mortality Mortality dilution, percent Ratio Percent Ratio Percent Ratio Percent 6. 25 17/20 85 4/20 1/10 10 12. 5 12/20 60 2/20 10 0/10 0 12 hours 10/20 50 0/20 0 0/10 0 Control 20/20 100 17/20 85 5/10 50 6. 25 2/20 10 1/20 5 0/10 0 12. 5 0/20 0 0/20 0 0/10 0 48 hours 25 0/20 0 0/20 0 0/10 0 Controls 19/20 95 12/20 60 4/10 40 3,031,378 Patented Apr. 24, 1962 EXAMPLE 2 "sodium glucuronate as the same method as previously described, and maintained for 1 hour at 22 C., following in an ice chest for 48 hours. After this procedure the virus suspensions were inoculated into mice intracerebrally Table 2: a

' Table 2 EXPERIMENTS ON INACTIVA'IION OF THE JAPANESE B ENOEPHALITIS VIRUS BY SODIUM GLUCURONATE Concentration Mortality of the sodium glucuronate solution used in dilution, Ratio Percent percent 6. 25 1/10 10 12. 5' 1/ 10 10 25 0/10 0 Control 9/10 EXAMPLE 3 in Table 3:

Table 3 EXPERIMENTS ON INACTIVATION OF THE INFLUENZA VIRUS \VITH SODIULI GLUCURONATE Concentration Morbidity of the sodium glucuronate solution used in dilution, Ratio Percent percent 6. 25 0/8 0 12.5 0/8 0 25 0/7 0 Control 3/7 43 EXAMPLE 4 10 10- and 10- dilutions of the rabies virus (CVS strain, i.c., LD =10* /0.2 ml.) were prepared in the same manner as in Example 1, were kept 1 hour at 22 C. and in an ice chest for 48 hours and were inoculated respectively into mice intracerebrally (0 dd strain 10 to 12 g.).

The results are given in Table 4:

Table 4 EXPERIMENTS ON INACTIVATION O F RABIES VIRUS WITH SODIUM GLUCURONATE Concentration of the rabies virus dilution Concentration of the sodium 10 10' 10- glucuronate solution used in dilution, Mortality Mortality Mortality percent Ratio Percent Ratio Percent Ratio Percent 6. 25 8/10 80 0/10 0 0/10 0 12. 5 0/10 0 0/10 0 0/10 0 25 0/10 0 0/10 0 0/10 0 Control 10/10 10/10 100 7/10 70 The results for 2 weeks after inoculation are given in III: M/SOO N-benzyl- References Cited in the file of this patent bodies in each group were tested by intracerebral inoculation by the conventional method.

Glucuronolactone (1952), Corn Products Sales Co.

sisting of glucuronolactone, glucuronic acid and alkali metal glucuronate and maintaining the pH at between 5-8.

Chem. Div., 17 Battery PL, NY. 4, N.Y., 20 pp.

IV: Heat-inactivated virus.

11: Formalinized virus.

From these experiments it is obvious that sodium glucuronate inactivates viruses in vitro. Likewise, glucuronic 7 days after the N-oxide hydrochloride inactivated virus.

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Testing method.-As in Example 5 (2) the mouse was inoculated with the above treated virus.

final immunization, the mice were sucrified and the sera were obtained respectively. Titer of neutralizing anti- 

1. A METHOD FOR INACTIVATING SUSPENSIONS OF LIVE VIRUSES COMPRISING TREATING THE VIRUS SUSPENSION AT A TEMPERATURE OF LESS THAN 23*C. WITH A COMPOUND SELECTED FROM THE GROUP CONSISTING OF GLUCURONOLACTONE, GLUCURONIC ACID AND ALKALI METAL GLUCURONATE AND MAINTAINING THE PH AT BETWEEN 5-8. 